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1 Article 2 Biodiversity, evolution and ecological specialization 3 of baculoviruses: a treasure trove for future applied 4 research

5 Julien Thézé1,2; Carlos Lopez-Vaamonde1,3; Jennifer S. Cory4; Elisabeth A. Herniou1 6 1 Institut de Recherche sur la Biologie de l’Insecte, UMR 7261, CNRS - Université de Tours, 37200 Tours, France; 7 [email protected] 8 2 Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3SY, UK; [email protected] 9 3 INRA, UR633 Zoologie Forestière, 45075 Orléans, France; [email protected] 10 4 Department of Biological Sciences, Simon Fraser University, Burnaby, V5A 1S6, British Columbia, Canada; 11 [email protected] 12 13 * Correspondence: [email protected]; Tel.: +33-247-367381 14

15 Abstract: The Baculoviridae, a family of insect-specific large DNA viruses, is widely used in both 16 biotechnology and biological control. Its applied value stems from millions of years of evolution 17 influenced by interactions with their hosts and the environment. To understand how ecological 18 interactions, have shaped baculovirus diversification, we reconstructed a robust molecular 19 phylogeny using 217 complete genomes and ~580 isolates for which at least one of four 20 lepidopteran core genes was available. We then used a phylogenetic-concept-based approach 21 (mPTP) to delimit 165 baculovirus species, including 38 species derived from new genetic data. 22 Phylogenetic optimization of ecological characters revealed a general pattern of host conservatism 23 punctuated by occasional shifts between closely related hosts and major shifts between 24 lepidopteran superfamilies. Moreover, we found significant phylogenetic conservatism between 25 baculoviruses and the type of plant growth (woody or herbaceous) associated with their insect 26 hosts. In addition, we found that colonization of new ecological niches sometimes led to viral 27 radiation. These macroevolutionary patterns show that besides selection during the infection 28 process, baculovirus diversification was influenced by tritrophic interactions, explained by their 29 persistence on plants and interactions in the midgut during horizontal transmission. This complete 30 eco-evolutionary framework highlights the potential innovations that could still be harnessed from 31 the diversity of baculoviruses.

32 Keywords: cophylogeny; granulovirus; host shifts; Lepidoptera; mPTP; multitrophic interactions; 33 niche conservatism; nucleopolyhedrovirus; phylogenetics; resource tracking; species delimitation. 34

35 1. Introduction 36 The use of baculoviruses (BVs) as expression vectors has mainly focused on the development of 37 a single virus, namely Autographa californica multiple nucleopolyhedrovirus (AcMNPV) [1], and to a 38 lesser extent, Bombyx mori nucleopolyhedrovirus (BmNPV) [2]. The commercial success of AcMNPV 39 for biotechnological application is undeniable. Aside from historical reasons, the fact that it is a 40 generalist virus that can infect cell lines from different hosts probably explains why AcMNPV was 41 first chosen. The family Baculoviridae, however, encompasses hundreds of isolates, many of which 42 have been studied in the context of biological control of insect pests, but some of which could in the 43 future prove equally as useful as AcMNPV for biotechnological applications, by providing new 44 molecular and biochemical products with contrasting antigenic properties or by infecting new, more 45 productive cell lines. In this context, it is important to describe the taxonomical diversity of BVs to 46 ensure that new isolates developed for biotechnological products are not examples of commonly

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47 used viral species, which could infringe patents. However, to delineate species boundaries requires 48 an understanding of BV evolution based on both historical and ecological perspectives. 49 The delimitation of species is a notoriously difficult question in biology, particularly for those 50 organisms for which the biological species concept cannot be applied. Viral species are defined by 51 the International Committee on Taxonomy of Viruses (ICTV) as “a monophyletic group of viruses 52 whose properties can be distinguished from those of other species by multiple criteria” [3]. This 53 general definition recognizes the importance of shared evolutionary history to define viral lineages 54 but allows for other biological criteria to be used, whether or not they may have an evolutionary 55 meaning. Recent advances in virus discovery (e.g. [4]) have spurred the need to reconsider the 56 method used for virus classifications [5,6]. Genetic distances are commonly used to delimitate viral 57 species based on distance cut-offs [5]. In the case of BVs, the cut-off between species has been 58 defined as between 0.015 and 0.05 based on the Kimura-2-parameter on sequence alignments of the 59 polyhedrin, lef-8 or lef-9 genes [7]. There is no doubt about the usefulness of those distance 60 thresholds to cluster closely related isolates (distance below 0.015) and to separate distantly related 61 viruses. However, there is often a high degree of uncertainty when clustering at intermediate levels 62 of genetic distances (up to 0.05). Intermediate levels of genetic differentiation could be found within 63 widespread, generalist, or fast evolving viral species. In these cases, the sole use of genetic distances 64 is insufficient to infer clusters and the delimitation criteria are left to the judgment of the taxonomist. 65 There is therefore a need to consider alternative analyses for the delimitation of BV species, which 66 could also be applied to other viral families. Recent advances in molecular phylogenetics now allow 67 the clustering of sequences into species groups, taking into account differences in evolutionary rates 68 or based on coalescence theory (e.g. [8,9]), and which do not require prior knowledge of species 69 limits. These automated methods, designed for DNA barcoding (e.g. [10,11]) to group individual 70 animal sequences of typically one gene into species clusters, are based on universal theory and can 71 therefore be applied to other genes and taxa [12], including viruses. 72 As insect-specific, enveloped, rod-shaped viruses with large circular double-stranded DNA 73 genomes, BVs replicate in the nucleus of infected host cells. Their life cycle is characterized by the 74 packaging of virions within a matrix of protein, forming the so-called occlusion body (OB), which is 75 the vehicle of horizontal transmission. Over 600 BVs have been described from the insect orders 76 Lepidoptera, Hymenoptera and Diptera. More than 90% of the known BVs have been isolated from 77 lepidopteran hosts [13]. The current classification [14] divides the Baculoviridae family into four 78 genera [15]. Two genera, the Alphabaculovirus and Betabaculovirus, infect lepidopteran species, and 79 differ by the morphology of their OBs forming the nucleopolyhedroviruses (NPVs) and 80 granuloviruses (GVs) respectively. The OBs of NPVs contain many virions and have been isolated 81 from both lepidopteran and non-lepidopteran hosts; in contrast, the OBs of the lepidopteran GVs 82 contain a single virion [14]. Phylogenetic and comparative genomic studies have shown that both 83 Lepidoptera specific genera are sister groups and that they diversified during the Mesozoic Era after 84 the diversification of insect orders [7,16-18]. 85 OBs are the between host transmission stage of the virus and allow the virus to survive outside 86 the host [19]. BVs are generally obligate killers as the host must die before the OBs are released into 87 the environment for horizontal transmission. But latent baculovirus infections [20,21] and vertical 88 transmission [22-24] have been reported. Insect larvae become infected by ingesting host plant tissue 89 contaminated with OBs. Thus, plant and virus have a close interaction in the insect gut and plant 90 chemicals can interact with the virus in numerous ways resulting in altered infectivity [25]. There is 91 therefore the potential for plants to play an important role in the evolution of insect-BV interactions 92 [25-28]. A better understanding of these diverse evolutionary interactions might point to new 93 molecular targets that could lead to biotechnological innovations. 94 In this study our main aims were to 1) provide a robust phylogeny for the whole family 95 Baculoviridae using all the genetic data generated so far; 2) delineate baculovirus species using a 96 non-a-priori phylogenetic-concept-based approach; 3) study the evolutionary history of host use of 97 BVs, assessing the level of host specialization and role of host shifts in the speciation of BVs; 4)

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98 elucidate the role of host plants in the evolution of BVs. Our results shed light on the biodiversity 99 ecological and evolutionary factors that drive Baculoviridae diversification.

100 2. Materials and Methods

101 2.1 Virus isolate sequence database 102 We created a DNA sequence database containing sequences of at least one of the four 103 lepidopteran BV core genes, late expression factor 8 (lef-8), late expression factor 9 (lef-9), per os 104 infectivity factor 2 (pif-2) and polyhedrin (polh). We chose those genes because it has been shown 105 that they bear a strong phylogenetic signal [16] and have been abundantly sequenced. The database 106 includes sequences collated from the public databases GenBank, EMBL and DDBJ (version April 107 2018), and we tried to obtain sequences from one hundred historical BV samples originating from 108 the reference collection at the Centre for Ecology and Hydrology (NERC-CEH), Wallingford, United 109 Kingdom. Primers, PCR amplification protocols and Sanger sequencing are previously described 110 [7,15]. Following current taxonomic practice, each BV sample was associated with the host species 111 from which it was isolated and OB morphology (NPV or GV), as well as sampling information about 112 the virus isolate or strain (if provided), and the name of the first author of the study from which BV 113 sequences derived (Table S1).

114 2.2 Host ecology database 115 For each BV isolate studied, we associated host ecological data. We included the taxonomy of 116 each insect host species (superfamily, family, subfamily), and their geographical distribution 117 (ecozone), established from localities where insect hosts were observed in the field. We also included 118 the host plant range of each insect host, from which we determined the associated insect host plant 119 growth type, distinguishing woody perennial (including shrubby and suffrutescent plants) and 120 annual/biennial herbaceous plants. Information was mainly extracted from the database of the 121 world’s lepidopteran host plants of the Natural History Museum of London [29], from the Barcode 122 of Life Data System (BOLD) [30] and from the literature [31] (Table S2).

123 2.3 Baculovirus core-genome phylogeny 124 A phylogenomic approach was used to reconstruct the BV core-genome phylogeny. BVs 125 possess 37 core genes [32] identified in all completely sequenced genomes. Amino acid multiple 126 alignments were performed on the 37 BV core gene products using MAFFT program [33] and, 127 alignments were concatenated prior to the phylogenetic reconstruction. A maximum likelihood 128 (ML) phylogenetic inference was performed on the concatenated multiple alignment under the Le 129 and Gascuel amino acid substitution model, with a gamma distributed among site rate variation and 130 a proportion of invariant sites (LG + Γ + I), as determined by ProtTest3 [34]. The ML analysis was 131 performed with RAxMLv.8.2 program [35] and statistical support for nodes in the ML tree was 132 assessed using a bootstrap approach (with 100 replicates).

133 2.4 Baculovirus isolate phylogeny 134 For each of the four BV core genes (lef-8, lef-9, pif-2 and polh) codon-based multiple alignments 135 were performed on all BV isolate sequences. Alignments were then concatenated and completed 136 with gaps for isolates, for which up to three of the four lepidopteran core genes might be missing. A 137 ML phylogenetic inference was performed on the concatenated codon-based multiple alignment 138 with RAxMLv.8.2 using the BV core-genome phylogeny as backbone constraint tree. The BV isolate 139 phylogeny was reconstructed using the best-fitted substitution model and parameters GTR + Γ + I, as 140 determined by jModelTest2 and statistical support for nodes in the ML tree was assessed using a 141 bootstrap approach (with 100 replicates). 142

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143 2.5 Species delimitation 144 A species delimitation analysis was performed on the BV isolate phylogeny using a 145 phylogenetic-species-concept-based method, the multi-rate Poisson tree processes (mPTP) model 146 [36]. The mPTP method is an improved version of the PTP model [9], which models speciation or 147 branching events in terms of number of substitutions and uses heuristic algorithms to identify the 148 most likely classification of branches into population and species-level processes. Moreover, mPTP 149 is fast and incorporates different levels of intraspecific genetic diversity deriving from differences in 150 either the evolutionary history or sampling of each species [36]. The mPTP model delimit species on 151 phylogenies without a priori assumptions, instead of the genetic distance cut-off commonly used. 152 The mPTP results were compared to the commonly used genetic-distances-based method. A 153 previous study calculated an intra-species genetic distance of ≤ 0.015 (up to 0.05 with 154 complementary information) as marker to delimit BV species [7]. This distance is commonly used by 155 the BV taxonomists and was evaluated using the Geneious plugin SpDelim [37], which assesses the 156 within and between species genetic distances in a phylogenetic tree. For both mPTP and SpDelim 157 analyses, the BV isolate ML phylogeny was used.

158 2.6 Baculovirus species phylogeny 159 From the species delimitation analysis, one isolate per cluster was selected as a representative of 160 a putative BV species (e.g: isolates with a complete genome or isolates with the four core genes 161 sequenced) and singletons were considered as distinct putative BV species. The topology of these 162 species-representative isolates was extracted from the BV isolate phylogeny by pruning the other 163 taxa, using the ‘ape’ package [38] for R. A new amino acid multiple alignment was created with only 164 the set of the species-representative isolates. A Bayesian phylogenetic inference was performed on 165 this alignment using BEASTv.1.8.4 [39]. The substitution model and parameters LG + Γ + I were used 166 as well as a fixed strict clock of 1.0. The topology of the above species-representative isolate tree was 167 used as target tree for the calculation of the consensus tree and posterior probabilities.

168 2.7 Phylogeny-trait correlation and ancestral state estimations 169 Correlations between phylogenetic tree structure and isolate trait discrete values (see Table S2) 170 were assessed using the methods implemented in BaTS/befi-BaTS [40]. The number of state 171 randomizations was defined to 100 to yield a null distribution. A correlation was considered 172 unambiguously positive if the Association Index (AI), the Parsimony Score (PS), the Phylogenetic 173 Diversity (PD), the Nearest Taxa (NT) and the Net Relatedness (NR) indices, the Unique Fraction 174 (UniFrac) index, and the maximum monophyletic clade (MC) size probabilities were ≤ 0.01. 175 Phylogenetic uncertainty was taken into account by using the set of tree topologies estimated by the 176 above Bayesian phylogenetic inference. For those traits that obtained significant P-value, a new 177 Bayesian phylogenetic inference was performed on the species-representative isolate alignment with 178 the same set of parameters as above and adding discrete trait partitions to reconstruct ancestral 179 states.

180 2.8 Host-virus cophylogeny 181 To test for the existence of statistically significant topological congruence between BV species 182 and Lepidoptera hosts, we estimated the maximum number of cospeciation events at different 183 taxonomic levels (superfamily, family, subfamily and genus), needed for reconciling BV species and 184 host phylogenies in TreeMap 3b [41] and compared this estimate to the distribution of 185 corresponding values obtained by randomizing different Lepidoptera trees 100 times while keeping 186 BV-host associations unchanged [42]. Tests at superfamily, family and subfamily taxonomic levels 187 compared the whole BV species phylogeny to published Lepidoptera phylogenies [43-45]. Tests at 188 the genus level were performed by reconstructing lepidopteran host species phylogenies based on 189 published phylogenies (Noctuidae [46], Erebidae [47,48], Lasiocampidae [49], Spodoptera genus [50]) 190 or when not available on CO1 sequence data extracted from BOLD public database [30] and

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191 compared to the BV species tree simplified by pruning monophyletic clades of species sharing a 192 given host genus down to a single species. Analyses were performed using 25 starts from random 193 maps and heuristic searches for up to 25 generations.

194 3. Results

195 3.1. Baculovirus phylogeny 196 Here we present a comprehensive phylogeny of BVs, including most of the relevant BV genetic 197 data available to date. A data-mining analysis was performed on public genetic databases and 198 resulted in the collation of 749 BV isolates properly formatted, containing the nucleotide sequences 199 of at least one of four lepidopteran BV core genes (lef-8, lef-9, pif-2 and polh genes). We were also 200 able to determine the sequences for 45 historical isolates from our own collection. Overall our 201 working sequence database contains 2053 nucleotide sequences (564 lef-8 genes, 498 lef-9 genes, 283 202 pif-2 genes and 708 polh genes) belonging to 794 BV isolates (Table S1). A total of 235 isolates have 203 the sequences of the four genes. 204 Among the BV isolates, 217 have complete genomes and were used to reconstruct the highly 205 supported BV core-genome phylogeny (Figure S1), inferred from the concatenated multiple amino 206 acid alignment of the 37 BV core genes. The tree topology is in accordance with previous studies 207 showing four BV genera infecting three distinct insect orders: the genera Alphabaculovirus and 208 Betabaculovirus infect lepidopteran species, the genus Gammabaculovirus infects hymenopteran 209 species and the genus Deltabaculovirus infects dipteran species [14,15]. The topology obtained was 210 used as backbone constraint tree to reconstruct the BV isolate phylogeny. The BV isolate phylogeny 211 (Figure 1; Figure S2) is quite well supported and is in accordance with previous studies showing 212 similar topologies [7,14,17] with notably a separation of two major monophyletic subgroups of 213 alphabaculoviruses (Group I, Group II). In addition, four minor monophyletic subgroups are 214 outgroups to Group I and II. Within Group I we observed a clear division into two distinct 215 monophyletic clades (I.a and Ib) and Group II is divided into three distinct monophyletic clades (II.a, 216 II.b and II.c) (Figure 1; Figure S2).

217 3.2. Species delimitation 218 To infer macroevolutionary process, it is necessary to conduct analyses at the species level to 219 avoid interference from intraspecific diversity. A species delimitation analysis was performed on the 220 BV isolate tree (Figure 1; see also Figure S2) using the mPTP method and then compared to the 221 commonly used genetic-distances based method. The mPTP approach delimited 165 distinct 222 putative BV species of which 70 putative BV species have been derived from clusters of two or more 223 isolates and 95 putative species are singletons (based on a unique viral isolate) (Figure 1; see also 224 Figure S2 and Figure S3). The dataset includes 38 new baculovirus species from our historical 225 isolates. The genetic-distances based method delimited 178 putative BV species (72 clusters and 106 226 singletons; Figure 1; see also Figure S2 and Figure S4). The two approaches show 157 species in 227 common, the differences in the genetic-distances based method were mostly observed in 228 alphabaculovirus species showing high levels of sampling and genetic variability (Figure 1; see also 229 Figure S2). 230

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234

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235 Figure 1. Baculovirus isolate phylogeny. The tree was obtained from a maximum likelihood 236 inference analysis of the concatenated codon-based alignment (794 taxa) of four lepidopteran 237 baculovirus core genes with the baculovirus core-genome phylogeny used as backbone tree. External 238 clades colored in red correspond to clusters determined by both the mPTP and SpDelim species 239 delimitation analysis and in blue the clusters not determined by SpDelim. The two star symbols 240 points out the same node in the tree. Baculovirus isolates generated in this study are highlighted in 241 orange. Statistical support for nodes in the tree corresponds to bootstraps (with 100 replicates).

242 Within the 165 distinct species identified by mPTP, 116 putative species are alphabaculoviruses, 243 45 are betabaculoviruses, three are gammabaculoviruses and one is a deltabaculovirus. The 10th 244 report of the ICTV (2017, https://talk.ictvonline.org/ictv-reports/ictv_online_report/) only currently 245 recognizes 68 species in the Baculoviridae: 40 in the genus Alphabaculovirus, 25 in the genus 246 Betabaculovirus, two in the genus Gammabaculovirus and one in the genus Deltabaculovirus [14]. The 247 reduced tree containing only putative BV species (Figure 2) suggests, therefore, 97 new putative BV 248 species, including 76 alphabaculoviruses and 20 betabaculoviruses.

249 3.3. Phylogenetic conservatism and host shifts 250 Out of the 165 alphabaculovirus and betabaculovirus species identified by mPTP, 161 are 251 associated with 187 Lepidoptera species from 24 different families. For all those lepidopteran hosts, 252 we compiled a dataset with the taxonomy (superfamily, family and subfamily), the biogeographical 253 distribution and the insect host plant range, from which we determined the host plant growth type 254 (woody versus herbaceous) (Table S2). We performed phylogeny-trait correlation and ancestral state 255 estimation analyses on the BV species phylogeny to measure whether those traits have evolved 256 randomly or show phylogenetic conservatism. The different phylogeny-trait correlation tests show 257 unequivocal significant associations between the taxonomy of insect hosts, the insect host plant 258 growth type and the BV species phylogeny (AI, PS, PD, NT, NR, UniFrac and MC; P ≤ 0.01, Table 1). 259 In contrast, no significant association was found between the insect host biogeography distribution 260 and the BV species phylogeny, as shown by the high p-values of certain tests (NR, UniFrac and MC; 261 p > 0.01, Table 1). 262 263 Table 1. Phylogeny-trait correlations estimated under different statistical methods

Traits AI1 PS1 PD1 NT1 NR1 UniFrac1 MC2 Host superfamily <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 / 21 Host family <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 / 21 Host subfamily <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 / 7 Host biogeography <0.001 <0.001 <0.001 <0.001 0.58 0.029 0.019 / 4 distribution (ecozone) Insect host plant growth <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 / 22 type (Herb/Woody) 264 1 p-value of AI: association index; PS: parsimony score; PD: phylogenetic diversity index; NT: nearest taxa 265 index; NR: net relatedness index; UniFrac: unique fraction index; MC: maximum monophyletic clade size 266 probability; 2 p-value / size of MC: Maximum monophyletic clade size probability and size (number of taxa) of 267 the largest monophyletic clade associated to one character of the trait 268 269 Ancestral state estimations on the baculovirus species tree were performed on traits that 270 showed significant associations such as host taxonomy and insect host plant growth. The 271 estimations show highly significant levels of phylogenetic conservatism with the different host 272 taxonomic levels and the insect host plant growth type (Table 1). For the host taxonomy trait we 273 illustrate only the insect host superfamily optimization as it is the higher taxonomic level and has a 274 reduced number of character states (Figure 2). The ancestral state estimation of the insect host

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275 superfamilies shows that closely related BV species tend to infect closely related lepidopterans (BVs 276 cluster together according to the lepidopteran superfamilies) (Figure 2A). The host use optimization 277 identifies owlet moths (Noctuoidea) as the most likely ancestral hosts of BVs (Figure 2A). Despite 278 significant levels of phylogenetic conservatism several major host shifts across different 279 lepidopteran superfamilies have occurred during BV evolution. Thus, in the Betabaculovirus genus 280 we can see a host shift from Noctuoidea to the Tortricoidea superfamily and then colonization of 281 several superfamilies: Papilionoidea, Zygaenoidea, Bombycoidea and a shift back to the Noctuoidea 282 (Figure 2A). The Alphabaculovirus genus splits into two large lineages (Group I and II) and four small 283 lineages (Figure 2A). Group I colonizes several Lepidoptera superfamilies, Clade I.a shows no host 284 conservatism harboring a very diverse host range, whereas Clade I.b shows a medium level of host 285 conservatism with a shifting from Noctuoidea to Tortricoidea and Papilionoidea. Group II shows a 286 clear split between Clades II.a, II.b and II.c, with clades II.a and II.b showing a strong conservatism 287 towards Noctuoidea with Clade II.a being specific to Noctuoidea with no apparent host shift. Both 288 clades II.b and II.c show host shifts from Noctuoidea to Geometroidea and Lasiocampoidea and 289 colonization of Bombycoideae, Tortricoidea, Tineoidea and Papilionoidea (Figure 2A). 290 Insect host plant growth type (Figure 2B), which can be considered as the local habitat of the 291 virus, shows a strong phylogenetic conservatism (Table 1). Our analyses show woody plants as the 292 most likely ancestral ecological niche of the first lepidopteran BVs. The Betabaculovirus genus is 293 ancestrally associated with herbaceous plants before colonizing woody plants. The Alphabaculovirus 294 genus is ancestrally associated with woody plants. Group I shows a strong association with woody 295 plants with few shifts to herbaceous plants. The ancestors of group II likely fed on woody plants 296 with colonization of herbaceous plants (Clade II.a). The three small alphabaculovirus clades, that are 297 outgroups to Clade I and II, also show a shift to herbaceous plants. The most notable result on these 298 analyses is the long-term associations with a particular insect host plant growth type. Strikingly, we 299 noticed in the Group II of the Alphabaculovirus genus a split of virus infecting Noctuoidea, which 300 seems to be the result of the colonization of herbaceous plant local habitat in Clade II.a (Figure 2).

301 3.4. Cophylogeny between baculoviruses and their lepidopteran hosts 302 The association between the topology of the BV species phylogeny and that of their 303 lepidopteran hosts (at superfamily, family and subfamily levels) is not significantly different from 304 random. In contrast, we found significant topological congruence (P < 0.5) for six BV clades at 305 different taxonomic levels (see the six clades highlighted in Figure 2). The Alphabaculovirus genus 306 gathers all the clades with significant topological congruence with host topologies. One clade 307 infecting Tortricoidea hosts was detected in Group I, whereas four clades infecting Noctuoidea hosts 308 were identified, and notably three in Group II. One clade infecting Lasiocampoidea was also 309 detected in Group II. 310 311

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312

313 Figure 2. Phylogenetic optimizations on the lepidopteran baculovirus species tree. Optimizations 314 were obtained by ancestral state estimation of A) host Lepidoptera at superfamily level, and B) insect 315 host plant growth type traits on the ingroup of Lepidoptera infecting baculovirus. The term 316 “Herb/woody” in the legend means that insect hosts feed on herbaceous plants as well as woody 317 plants. Black circles close to phylogenetic nodes refer to posterior probabilities over 0.75. Baculovirus 318 species highlighted with black line rectangles correspond to clades that are significantly 319 topologically congruent (P < 0.5) with lepidopteran host clades.

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320 4. Discussion

321 4.1. Reconstructing Phylogenies and Delineating Species 322 The Baculoviridae is by far the best described insect DNA virus family. For the last 50 years, BV 323 use in biotechnology, either as expression vectors [1] or as microbial control agents for insect pests 324 [51], has led to a vast production of molecular data, especially for the genera infecting the 325 Lepidoptera, on which this study is focused. The first objective of this study was to reconstruct the 326 most accurate and exhaustive BV isolate phylogeny in order to set a solid framework for species 327 delimitation and macroevolutionary inference. 328 The backbone of the tree was built based on 217 whole BV genomes (Figure S1). A secondary 329 ML analysis included an additional 577 isolates, from which we obtained our isolate tree (Figure 1; 330 Figure S2). We chose to cluster these viral isolates into species, which are the basis of biological 331 classification, in order to study baculovirus speciation at a macroevolutionary scale. Baculoviruses 332 like most of large DNA viruses are slow-evolving viruses with an approximate mutation rate of 333 10-6/10-7 (expressed as the number of substitutions per nucleotide per generation, defined as a cell 334 infection in viruses) [52]. This mutation rate approaches those observed in Bacteria and lower 335 Eukaryotes. Consequently, we decided to use a phylogenetic-species-concept-based clustering 336 approach, the mPTP model [36], to delimit BV species and to compare the results to the commonly 337 used genetic-distances-based method (intra-species distance ≤0.015; up to 0.05 with complementary 338 information [7]). Our species delimitation results were consistent results with the current taxonomy 339 proposed by the ICTV [14]. Out of 165 BV species we were able to characterize 97 that are not yet 340 included in the ICTV report. The BV species phylogeny reflects current knowledge on BV diversity 341 and phylogenetic relationships (Figure 1; Figure2). Furthermore, this study is the first to use a 342 phylogenetic clustering approach for species delimitation in viruses, showing that its utility goes 343 beyond vertebrates [53], invertebrates [54,55] and bacteria [12] taxa. Moreover, this approach for 344 species delimitation fully respects the phylogenetic species concept and is less arbitrary than 345 commonly used genetic distance approaches, which do not take into account differences in 346 molecular evolutionary rates or sampling proportions and may thus vary depending on the biology 347 of the lineage studied and on the gene used for phylogenetic reconstruction. Nevertheless, both 348 species delimitation approaches gave relatively consistent results with an overlap of 157 species 349 determined by both methods. The few differences in the genetic distances-based method were 350 mostly observed in heterogeneous species clusters, with isolates infecting one or several hosts and 351 showing high genetic diversity typically resulting in clusters with genetic distances between 0.015 352 and 0.050 [7]. The phylogenetic-based approach does not contradict the results of the 353 genetic-distances based approach but gives additional information to resolve the uncertainties of the 354 genetic-distances based approach.

355 4.2. Evolution of host use and taxa sampling 356 Most of the BV species identified in our study have isolates that infect only one lepidopteran 357 host species. Strikingly, we generally found that closely related hosts belonging to the same genus 358 were infected by different viral species (for example in the genera Spodoptera, Lymantria, or 359 Malacosoma). This leads us to question the ecological reality and biological meaning of some isolates, 360 such as Trichoplusia ni NPV and Busseola fusca NPV within the Helicoverpa armigera NPV clade 361 (Figure 1). However, generalist viruses, capable of infecting different host species, belonging or not 362 to the same genus, exist and the hosts they infect generally have overlapping ecological niches (same 363 host plants). As an example, several isolates from different species of nymphalid butterflies that feed 364 on nettles (Urtica dioica) form a single alphabaculovirus species Vanessa atalanta NPV. 365 As parasites replicating exclusively in host cells, BVs are involved in durable and intimate 366 obligate interactions with their host, implicating long-term coevolution. The phylogenetic 367 conservation results suggest an ancestral and frequent association with hosts of the Noctuoidea 368 superfamily. This possibly could reflect the actual evolution of lepidopteran BVs and their current 369 host range, as with ~42,000 out of ~157,000 described lepidopteran species the Noctuoidea is the

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370 most diversified superfamily (in comparison, the second most abundant superfamily is the 371 Geometroidea with ~23,000 species) [31]. Yet, BV genetic data from public databases is clearly biased 372 towards agro-economically important lepidopteran pests, characterized by large populations, 373 important for sustaining BV populations. The addition of new BV samples from our collection tends 374 to reduce the bias for pests, but BVs from pests still dominate our taxa sampling as these viruses 375 have been isolated and resequenced many times (i.e. large clusters of Cydia pomonella GV or 376 Helicoverpa armigera NPV; Figure 1) and as at least 62 out of 161 putative lepidopteran BV species 377 are associated with pests (Figure 2A; Table S2). As numerous lepidopteran pests belong to the 378 Noctuoidea superfamily, this probably increases the representation of Noctuoidea infecting BVs in 379 our dataset and could have biased our phylogenetic conservatism results (77 out of 161 putative 380 lepidopteran BV species analyzed attack Noctuoidea, Figure 2A). Only a diverse BV sampling, more 381 representative of the lepidopteran diversity could confirm if Noctuoidea played a key role in BV 382 evolution.

383 4.3. Cophylogeny and host shifts 384 Cophylogenetic analyses show no topological congruence between Lepidoptera and the BV 385 species tree, but do show significant cophylogenetic signal between certain internal nodes of the BV 386 species phylogeny and the associated insect host species nodes (Figure 2). This means that present 387 host-use patterns in BVs result mainly from a pattern of host conservatism punctuated with 388 occasional shifting among pre-existing insect lineages [56,57]. This coevolutionary association 389 concurs with a general process of colonization by host tracking [58,59] as previously suggested at the 390 macroevolutionary level of several insect DNA virus families [18]. However, this type of 391 coevolutionary association is only observable for crown groups of viruses infecting hosts belonging 392 to the same family and is lost in more basal nodes. Indeed, over a short timeframe BV speciation 393 remains intimately linked to the speciation of their host, following the biogeography of their hosts 394 and ultimately in certain lineages we observed BV phylogenies that are the mirror images of their 395 host’s. Yet on a larger evolutionary scale, the insect-host coevolutionary relationship signal is 396 confused, strongly suggesting that other factors act on BV evolution.

397 4.4. Ecological Specialization 398 The distinctive feature of the BV life cycle compared to most viruses is that they produce a 399 transmission stage, which persists outside of the host and has the ability to resist environmental 400 degradation. This feature is also found in other insect viruses such as entomopoxviruses and 401 cypoviruses, which have similar life cycles and OBs. This highlights the importance and the 402 persistence of this dissemination process in insect viruses [60]. As BVs only infect larvae and need to 403 be ingested to initiate infection, they have an intimate association with the plants that their hosts 404 feed on. In addition, there is an increasing body of evidence showing that host plant chemistry can 405 moderate the BV infection process [25,28]. Thus host plant characteristics could define the local 406 biotopes of BVs. We therefore searched for conservatism with the type of plant used by insect hosts, 407 distinguishing woody perennial (including shrubby and suffrutescent plants) and herbaceous 408 plants. Results show ancestral associations to particular plant groups over several tens of million 409 years (Figure 2B), suggesting a pattern of plant-use conservatism punctuated with sporadic shifts 410 between plant growths. This underlines the predominant role of host plant association in BV 411 evolution. As a consequence, BV diversification entangles patterns of host and local biotope 412 conservatism. 413 Virus ecological niches are considered in general as defined only by their hosts, 414 underestimating other factors and notably the influence of the environment. The BV niche combines 415 a set of insect and insect host plant biotic conditions. Viruses consume the resources provided by 416 their hosts in a host tracking fashion and consequently are influenced by their geographical 417 distributions; hosts are therefore the set of biotic conditions where primary speciation takes places 418 through adaptation to insect immunity and competition with other parasites. Host spectrum and 419 hosts shifts are contained within local environments represented by a group of host plants. Host

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420 plant-use therefore defines a second ring of biotic environment that BVs experience. The association 421 with particular types of plants persists over millions of years and drives BVs towards particular 422 insect hosts. This is strikingly observable for the group II of the Alphabaculovirus genus, where BV 423 infecting Noctuoidea species are split in two distinct clades, in one clade (clade IIa) Noctuoidea 424 species are associated with herbaceous plants and in the other clade (clade IIb) they are associated 425 with woody plants (Figure 2). 426 At the level of the evolutionary history of BVs, our study points out the specialization of BVs 427 highlighted with topological congruence of certain BV-hosts associations. At the macroevolutionary 428 timescale, BVs are specialized to a particular insect order and notably to three orders of 429 holometabolous insects [15]. The BVs infecting Lepidoptera separate into two major groups with 430 different types of OBs, the NPVs and the GVs. GVs (Betabaculovirus) seem to be ancestrally 431 associated to herbaceous plants and to have colonized woody plants later. Strikingly, some GVs are 432 associated with internal feeding hosts such as the codling moth, Cydia pomonella, the potato tuber 433 moth, Phthorimaea operculella or the tea leaf-roller micromoth Caloptilia theivora and these types of 434 hosts are not infected by NPVs. The morphology of GV OBs, which are much smaller than those of 435 NPVs, may confer a more targeted dispersal strategy to increase the likelihood of reaching hosts 436 concealed within the plant. Therefore, it is predicted that leaf-mining and/ or stem-boring 437 Lepidoptera should more likely be infected by GVs than by NPVs. 438 NPVs (Alphabaculovirus) appear to be ancestrally associated to woody plants and to have 439 colonized herbaceous plants afterwards. NPVs belonging to group II are often very specialized to 440 their hosts according to topological congruence with their associated hosts and the clear split of 441 herbaceous (clade IIa) and woody (clade IIb) clades. By contrast, NPVs belonging to group I show a 442 complex pattern of associations to several lepidopteran superfamilies on herbaceous and woody 443 plants, suggesting more frequent host switches than those observed for group II or GVs. 444 Remarkably, this group includes the well known Autographa californica MNPV, which has a large 445 host range, spanning different Lepidoptera species belonging to distantly related families [61] 446 (Figure 1; Figure S2). This BV is a generalist virus, which is an uncommon trait in BVs. Generalism 447 could favor host switching and explain loss of BV-host phylogenetic congruence in certain BV 448 lineages, notably in the clade Ia of the Alphabaculovirus genus. 449 The host is by far the most important component of virus ecological niches, but not the only 450 one. Most previous studies focused on microevolutionary patterns of diversification, resulting in 451 host distribution outcomes to explain virus dynamics [62,63]. Here, we discuss the 452 macroevolutionary patterns of an entire virus family based on a reconstruction, as exhaustive as 453 possible, of its history. BVs have a peculiar life cycle where insect hosts and their associated plants 454 are entangled. Viral transmission and fitness are increased with the typical production of OBs for 455 environmental dissemination combined with the modification of host behavior, like the 456 enhancement of climbing behavior [64,65]. Plants are therefore the vessel of viral transmission. 457 Plants attacked by many lepidopteran species could support the evolution of generalist viruses. This 458 in turn could promote host shifts with the subsequent specialization of particular viral lineages. 459 Conversely plants attacked by few specialist Lepidoptera should foster the evolution of specialist 460 viruses. Multitrophic interactions have thus shaped BV evolution as shown by the combined 461 patterns of insect host and insect host plant conservatism, punctuated by occasional shifts among 462 pre-existing insect lineages. However, the direct evolutionary interactions between plants and BVs 463 remain undetermined.

464 5. Conclusion 465 Our current understanding of baculovirus diversity and evolution has been fuelled by decades 466 of research on biological control and biotechnological applications. Here we presented a complete 467 evolutionary framework of the known baculovirus diversity highlighting the complex ecological 468 interactions of these viruses with their hosts. Our study is the first to use a phylogenetic clustering 469 approach for species delimitation in viruses characterizing many new baculovirus species. Our 470 analyses show that host shifts played a major role in the diversification of baculoviruses. It also

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471 shows that the colonization of a new ecological niche (herbaceous plants) lead to the radiation of 472 some baculovirus lineages. The species richness resulting from millions of years of evolutionary 473 interactions between the host plant ecology/chemistry and the physiology and ecology of both 474 viruses and their associated insect hosts potentially still hides a treasure trove of genes and 475 molecules that could lead to innovative biotechnology.

476 Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: Baculovirus 477 core-genome phylogeny, Figure S2. Raw baculovirus isolate phylogeny, Table S1. Baculovirus isolate sequence 478 database, Table S2. Host ecology database. 479 Author Contributions: Conceptualization, Carlos Lopez-Vaamonde, Jennifer S Cory and Elisabeth A Herniou; 480 Formal analysis, Julien Thézé; Funding acquisition, Elisabeth A Herniou; Investigation, Julien Thézé and 481 Elisabeth A Herniou; Methodology, Julien Thézé and Carlos Lopez-Vaamonde; Resources, Jennifer S Cory; 482 Supervision, Jennifer S Cory and Elisabeth A Herniou; Writing – original draft, Julien Thézé and Elisabeth A 483 Herniou; Writing – review & editing, Carlos Lopez-Vaamonde and Jennifer S Cory. 484 Funding: The European Research Council Grant ERC-Stg 205206 GENOVIR supported this work. 485 Acknowledgments: The Centre for Ecology and Hydrology (NERC-CEH), Wallingford, United Kingdom is the 486 continued repository of historical viral isolates described in this paper 487 Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the 488 study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision 489 to publish the results.

490 References

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